The present invention is directed to compounds of the formula 
and pharmaceutically acceptable salts thereof. As used in formula I, and throughout the specification, the symbols have the following meanings:
R1=R2, COR3, CONH2, CONR2R3, COOR3, or SO2R3;
R2=H, alkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, heterocycloalkylalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl, or heteroarylium;
R3=alkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, heterocycloalkylalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl; 
xe2x80x83where i, j=0 or 1 but cannot both be 1, and Y=optionally substituted ethylene, alkene, alkyne, or any 2 adjacent carbon atoms of a cycloalkyl or cycloheteroalkyl ring of 3-7 atoms;
R4=alkyl of 2 or more carbon atoms, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, heterocycloalkylalkyl, alkyloxycarbonyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl, aryloxycarbonyl, or R9, provided that R4 is other than an optionally substituted phenyl, pyridyl, pyrimidyl, pyrazinyl, pyridazinyl or piperidinyl ring;
R5, R6, R7, R8=independently H, alkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, heterocycloalkylalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl, halo, or hydroxy, alkoxy, amino, NR15R16, thio, or alkylthio with the proviso that only one such heteroatom group is bonded to any one carbon atom; 
xe2x80x83where Z=O, NR12, or S;
R10, R11=independently H, alkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, heterocycloalkylalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl, halo, hydroxy, alkoxy, alkylcarbonyloxy, carboxy, alkyloxycarbonyl, amino, NR13R14, carbamoyl, ureido, thio, or alkylthio; and
R12, R13, R14, R15, and R16=independently H, alkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, heterocycloalkylalkyl, aryl, heteroaryl, arylalkyl, or heteroarylalkyl.
The compounds of formula I are protein kinase inhibitors and are useful in the treatment of proliferative diseases, for example, cancer, inflammation and arthritis. They may also be useful in the treatment of Alzheimer""s disease, and cardiovascular disease.
The present invention provides for compounds of formula I, pharmaceutical compositions employing such compounds, and for methods of using such compounds.
Listed below are definitions of various terms used to describe the compounds of the instant invention. These definitions apply to the terms as they are used throughout the specification (unless they are otherwise limited in specific instances) either individually or as part of a larger group.
It should be noted that any heteroatom with unsatisfied valences is assumed to have hydrogen atoms sufficient to satisfy the valences.
Carboxylate anion refers to a negatively charged group xe2x80x94COOxe2x88x92.
The term xe2x80x9calkylxe2x80x9d or xe2x80x9calkxe2x80x9d refers to a monovalent alkane (hydrocarbon) derived radical containing from 1 to 12 carbon atoms unless otherwise defined. An alkyl group is an optionally substituted straight, branched or cyclic saturated hydrocarbon group. When substituted, alkyl groups may be substituted with up to four substituent groups, R as defined, at any available point of attachment. When the alkyl group is said to be substituted with an alkyl group, this is used interchangeably with xe2x80x9cbranched alkyl groupxe2x80x9d. Exemplary unsubstituted such groups include methyl, ethyl, propyl, isopropyl, n-butyl, t-butyl, isobutyl, pentyl, hexyl, isohexyl, heptyl, 4,4-dimethylpentyl, octyl, 2,2,4-trimethylpentyl, nonyl, decyl, undecyl, dodecyl, and the like. Exemplary substituents may include but are not limited to one or more of the following groups: halo (such as F, Cl, Br, I), haloalkyl (such as CCl3 or CF3), alkoxy, alkylthio, hydroxy, carboxy (xe2x80x94COOH), alkyloxycarbonyl (xe2x80x94COOR), alkylcarbonyloxy (xe2x80x94OCOR), amino (xe2x80x94NH2), carbamoyl (xe2x80x94NHCOORxe2x80x94 or xe2x80x94OCONHRxe2x80x94), urea (xe2x80x94NHCONHRxe2x80x94), amidinyl (xe2x80x94CNHNHR or xe2x80x94CNRNH2), or thiol (xe2x80x94SH). Alkyl groups as defined may also comprise one or more carbon to carbon double bonds or one or more carbon to carbon triple bonds.
The term xe2x80x9calkenylxe2x80x9d refers to a hydrocarbon radical straight, branched or cyclic containing from 2 to 12 carbon atoms and at least one carbon to carbon double bond.
The term xe2x80x9calkynylxe2x80x9d refers to a hydrocarbon radical straight, branched or cyclic, containing from 2 to 12 carbon atoms and at least one carbon to carbon triple bond.
The term xe2x80x9cethylenexe2x80x9d refers to a xe2x80x94CH2CH2xe2x80x94 group.
Cycloalkyl is a specie of alkyl containing from 3 to 15 carbon atoms, without alternating or resonating double bonds between carbon atoms. It may contain from 1 to 4 rings. Exemplary unsubstituted such groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc. Exemplary substituents include one or more of the following groups: halogen, alkyl, alkoxy, alkyl hydroxy, amino, nitro, cyano, thiol and/or alkylthio.
The terms xe2x80x9calkoxyxe2x80x9d or xe2x80x9calkylthioxe2x80x9d, as used herein, denote an alkyl group as described above bonded through an oxygen linkage (xe2x80x94Oxe2x80x94) or a sulfur linkage (xe2x80x94Sxe2x80x94), respectively.
Sulfoxide and sulfone denote groups bonded by xe2x80x94SOxe2x80x94 or xe2x80x94SO2xe2x80x94 linkages, respectively.
The term xe2x80x9calkyloxycarbonylxe2x80x9d, as used herein, denotes an alkoxy group bonded through a carbonyl group. An alkoxycarbonyl radical is represented by the formula: xe2x80x94C(O)OR, where the R group is a straight or branched C1-6 alkyl group.
The term xe2x80x9calkylcarbonylxe2x80x9d refers to an alkyl group bonded through a carbonyl group.
The term xe2x80x9calkylcarbonyloxyxe2x80x9d, as used herein, denotes an alkylcarbonyl group which is bonded through an oxygen linkage.
The term xe2x80x9carylalkylxe2x80x9d, as used herein, denotes an aromatic ring bonded to an alkyl group as described above.
The term xe2x80x9carylxe2x80x9d refers to monocyclic or bicyclic aromatic rings, e.g. phenyl, substituted phenyl and the like, as well as groups which are fused, e.g., naphthyl, phenanthrenyl and the like. An aryl group thus contains at least one ring having at least 6 atoms, with up to five such rings being present, containing up to 22 atoms therein, with alternating (resonating) double bonds between adjacent carbon atoms or suitable heteroatoms. Aryl groups may optionally be substituted with one or more groups including, but not limited to halogen, alkyl, alkoxy, hydroxy, carboxy, carbamoyl, alkyloxycarbonyl, nitro, trifluoromethyl, amino, cycloalkyl, cyano, alkyl S(O)m(m=O, 1, 2), or thiol.
The term xe2x80x9cheteroarylxe2x80x9d refers to a monocyclic aromatic hydrocarbon group having 5 or 6 ring atoms, a bicyclic aromatic group having 8 to 10 atoms, or a tricyclic aromatic group having 10 to 16 atoms, containing at least one heteroatom, O, S, or N, in which a carbon or nitrogen atom is the point of attachment, and in which one or two carbon atom(s) is optionally replaced by a heteroatom selected from O or S, or in which from 1 to 3 additional carbon atoms are optionally replaced by nitrogen heteroatoms, said heteroaryl group being optionally substituted as described herein. Exemplary heteroaryl groups include the following: thienyl, furyl, pyrrolyl, pyridinyl, imidazolyl, pyrrolidinyl, piperidinyl, thiazolyl, oxazolyl, triazolyl, pyrazolyl, isoxazolyl, isothiazolyl, pyrazinyl, tetrazolyl, pyridazinyl, pyrimidinal, triazinylazepinyl, indolyl, isoindolyl, quinolinyl, isoquinolinyl, benzothiazolyl, benzoxazolyl, benzimidazolyl, benzoxadiazolyl, benzofurazanyl and tetrahydropyranyl. Exemplary substituents may include, but are not limited to, one or more of the following: halogen, alkyl, alkoxy, hydroxy, carboxy, carbamoyl, alkyloxycarbonyl, trifluoromethyl, cycloalkyl, nitro, cyano, amino, alkylS(O)m(m=0, 1, 2), or thiol.
The term xe2x80x9cheteroaryliumxe2x80x9d refers to heteroaryl groups bearing a quaternary nitrogen atom and thus a positive charge.
The term xe2x80x9cheterocycloalkylxe2x80x9d refers to a cycloalkyl group (nonaromatic) in which one of the carbon atoms in the ring is replaced by a heteroatom selected from O, S or N, and in which up to three additional carbon atoms may be replaced by said heteroatoms.
The term xe2x80x9cquaternary nitrogenxe2x80x9d refers to a tetravalent positively charged nitrogen atom including, e.g. the positively charged nitrogen in a tetraalkylammonium group (e.g. tetramethylammonium, N-methylpyridinium), the positively charged nitrogen in protonated ammonium species (e.g. trimethylhydroammonium, N-hydropyridinium), the positively charged nitrogen in amine N-oxides (e.g. N-methyl-morpholine-N-oxide, pyridine-N-oxide), and the positively charged nitrogen in an N-amino-ammonium group (e.g. N-aminopyridinium).
The term xe2x80x9cheteroatomxe2x80x9d means O, S or N, selected on an independent basis.
The term xe2x80x9chalogenxe2x80x9d or xe2x80x9chaloxe2x80x9d refers to chlorine, bromine, fluorine or iodine.
When a functional group is termed xe2x80x9cprotectedxe2x80x9d, this means that the group is in modified form to preclude undesired side reactions at the protected site. Suitable protecting groups for the compounds of the present invention will be recognized from the present application taking into account the level of skill in the art, and with reference to standard textbooks, such as Greene, T. W. et al., Protective Groups in Organic Synthesis, Wiley, N.Y. (1991).
Suitable examples of salts of the compounds according to the invention with inorganic or organic acids are hydrochloride, hydrobromide, sulfate, phosphate. Salts which are unsuitable for pharmaceutical uses but which can be employed, for example, for the isolation or purification of free compounds I or their pharmaceutically acceptable salts, are also included.
All stereoisomers of the compounds of the instant invention are contemplated, either in admixture or in pure or substantially pure form. The definition of the compounds according to the invention embraces all possible stereoisomers and their mixtures. It very particularly embraces the racemic forms and the isolated optical isomers having the specified activity. The racemic forms can be resolved by physical methods, such as, for example, fractional crystallization, separation or crystallization of diastereomeric derivatives or separation by chiral column chromatography. The individual optical isomers can be obtained from the racemates by conventional methods, such as, for example, salt formation with an optically active acid followed by crystallization.
All configurational isomers of compounds of the present invention are contemplated, either in admixture or in pure or substantially pure form. The definition of compounds of the present invention very particularly embraces both cis (Z) and trans (E) alkene isomers, as well as cis and trans isomers of cycloalkyl or heterocycloalkyl rings.
Furthermore, with regard to the object compound I, it is to be understood that the compound includes tautomeric isomers. That is the compound of formula I may exist in either of the the following forms: 
in a state of equilibrium.
It should be understood that solvates (e.g. hydrates) of the compounds of formula I are also within the scope of the present invention. Methods of solvation are generally known in the art. Accordingly, the compounds of the instant invention may be in the free or hydrate form, and may be obtained by methods exemplified by the following schemes. 
As illustrated in Scheme I, a compound of formula III may be prepared by treating a solution of 5-nitro-3-pyrazole carboxylic acid (II) in absolute ethanol with H2SO4 to form the ester. The compound of formula IV may be prepared by treating nitro compound III with a reducing agent such as hydrogen in the presence of a catalyst such as palladium on charcoal, or with a metal reducing agent such as tin or iron. The amino group and one of the pyrazole ring nitrogen atoms are protected by treating a compound of formula IV with di-tert butyl dicarbonate or a comparable carbamylating agent and a base such as pyridine. The compond of formula V may be reduced with lithium aluminum hydride or a comparable reducing agent to give the alcohol VI, which may be oxidized by manganese dioxide or a comparable oxidizing agent to give an aldehyde of formula VII. The aldehyde of formula VII may be reacted with a phosphonate ester and a base such as potassium t-butoxide to provide an olefin of formula VIII. The olefin of formula VIII may be either cis (Z), trans (E) or a mixture of the two isomers. In the event that the compound of formula VIII is a single isomer, it may be equilibrated with its geometric olefin isomer by treatment with an agent such as ultraviolet light (UV) and acetone. In the specific instance illustrated the E alkene isomer VIII is interconverted with the Z alkene isomer IX. A compound of formula IX may be deprotected by treating with an acid such as trifluoroacetic acid or hydrochloric acid in a solvent such as dioxane to give the free amine X, which may be reacted with an acylating agent such as an isocyanate, acid anhydride, acid chloride, or the like, to give a compound of formula XI, which is a compound of formula I where R1 is CONR2R3 and Y is Z alkenyl. 
According to Scheme II, a solution of 5-ethylcarboxylate-3-aminopyrazole IV, and a base such as pyridine may be reacted with an acid chloride such as 2-methyl butanoyl chloride to give the compound of formula XII. Treatment of a compound of formula XII with a reducing agent such as lithium aluminum hydride (LAH) in tetrahydrofuran (THF) gives the alcohol of formula XIII, which may be oxidized by an oxidant such as manganese dioxide in acetone to give an aldehyde of formula XIV. Aldehydes of formula XIV may be treated with a phosphonate ester in the presence of a base such as potassium tert-butoxide to give compounds of formula XV. Analogous to Scheme I, compounds of formula XV(which is a compound of formula I where Y is an E alkene) may be treated with ultraviolent light and acetone to give a compound of formula XVI (which is a compound of formula I where R1 is COR3 and Y is Z alkenyl).
Additional compounds of formula I where Y is an alkyne may be prepared by methods known to those of ordinary skill in the art. A compound of formula I where Y is an alkene may be reacted with a reducing agent such as hydrogen in the presence of a catalyst such as palladium on carbon to provide a compound of formula I where Y is ethylene. Compounds of formula I where Y is an alkyne may be prepared from the compound of formula I where Y is an alkene by methods known in the art, such as halogenation and dehydrohalogenation.
The starting compounds of Schemes I and II are commercially available or may be prepared by methods known to one of ordinary skill in the art.
The compounds shown in Schema I and II are illustrative, and the methods described may be used by those of ordinary skill in the art to prepare the analogous intermediates encompassed by the genus. All compounds of formula I may be prepared by modification of the procedures described herein.
A first group of preferred compounds of formula I are those where:
R1=R2, COR3, or CONR2R3;
R2=H, alkyl, aryl, heteroaryl, arylalkyl, or heteroarylalkyl;
R3=alkyl, aryl, heteroaryl, arylalkyl, or heteroarylalkyl; 
xe2x80x83where i=j=0;
Y=optionally substituted ethylene, alkene, or any two adjacent carbon atoms of a cycloalkyl ring;
R4=alkyl of two or more carbon atoms, aryl, heteroaryl, or R9;
R5, R6, R7, R8=independently H, or alkyl; 
xe2x80x83where Z=O; and
R10, R11=independently H, or alkyl.
A second group of preferred compounds of formula I are those where:
R1=CONR2R3;
R2=H, alkyl, heteroaryl, arylalkyl, or heteroarylalkyl;
R3=aryl; 
xe2x80x83where i, j=0 or 1 but cannot both be 1, and Y=an optionally substituted ethylene or alkene;
R4=R9;
R5, R6, R7, R8=independently H, or alkyl; 
xe2x80x83where Z=O; and
R10, R11=independently H, or alkyl.
A third group of preferred compounds of formula I are those where:
R1=R2;
R2=alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, arylalkyl, or heteroarylalkyl; 
xe2x80x83where i, j=0 or 1 but cannot both be 1, and Y=an optionally substituted ethylene or alkene;
R5, R6, R7, R8=independently H, or alkyl;
R4=R9; 
xe2x80x83where Z=O;
R10=alkyl; and
R11=H.
A fourth group of preferred compounds of formula I are those where:
R1=COR3;
R3=alkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, heterocycloalkylalkyl, aryl, heteroaryl, arylalkyl, or heteroarylalkyl; 
xe2x80x83where i, j=0 or 1 but cannot both be 1, and Y=an optionally substituted ethylene, alkene or alkyne;
R4=R9;
R5, R6, R7, R8=independently H, or alkyl; 
xe2x80x83where Z=O;
R10=alkyl; and
R11=H.
Formula I compounds particularly useful in the methods of this invention include:
N,N-[(Z)-5-(2-(5-(1,1-dimethyl(ethyl))-oxazol-2-yl)vinyl)pyrazol-3-yl]2,6-difluorophenylaminocarbonyl amine;
N-[5-(2-(5-(1,1-dimethyl(ethyl))-oxazol-2-yl)vinyl)pyrazol-3-yl]2-(methyl)propanamide;
N-(Z)-[5-(2-(5-(1,1-dimethyl(ethyl))-oxazol-2-yl)vinyl)pyrazol-3-yl]2-(methyl)propanamide; and
N,N-[(E)-5-(2-(5-(1,1-dimethyl(ethyl))-oxazol-2-yl)vinyl)pyrazol-3-yl]2,6-difluorophenylaminocarbonyl amine, among others.
The compounds according to the invention have pharmacological properties; in particular, the compounds of formula I are inhibitors of protein kinases such as the cyclin dependent kinases (cdks), for example, cdc2 (cdk1), cdk2, and cdk4. The novel compounds of formula I are expected to be useful in the therapy of proliferative diseases such as cancer, inflammation, arthritis, Alzheimer""s disease and cardiovascular disease. These compounds may also be useful in the treatment of topical and systemic fungal infections.
More specifically, the compounds of formula I are useful in the treatment of a variety of cancers, including (but not limited to) the following:
carcinoma, including that of the bladder, breast, colon, kidney, liver, lung, ovary, pancreas, stomach, cervix, thyroid, prostate, and skin;
hematopoietic tumors of lymphoid lineage, including acute lymphocytic leukemia, B-cell lymphoma, and Burkett""s lymphoma;
hematopoietic tumors of myeloid lineage, including acute and chronic myelogenous leukemias and promyelocytic leukemia;
tumors of mesenchymal origin, including fibrosarcoma and rhabdomyosarcoma; and
other tumors, including melanoma, seminoma, teratocarcinoma, osteosarcoma, neuroblastoma and glioma.
Due to the key role of cdks in the regulation of cellular proliferation in general, inhibitors could act as reversible cytostatic agents which may be useful in the treatment of any disease process which features abnormal cellular proliferation, e.g., neuro-fibromatosis, atherosclerosis, pulmonary fibrosis, arthritis, psoriasis, glomerulonephritis, restenosis following angioplasty or vascular surgery, hypertrophic scar formation, inflammatory bowel disease, transplantation rejection, angiogenesis, and endotoxic shock.
Compounds of formula I may also be useful in the treatment of Alzheimer""s disease, as suggested by the recent finding that cdk5 is involved in the phosphorylation of tau protein (J. Biochem, 117, 741-749 (1995)).
Compounds of formula I may also act as inhibitors of other protein kinases, e.g., protein kinase C, her2, rafI, MEK1, MAP kinase, EGF receptor, PDGF receptor, IGF receptor, PI3 kinase, wee I kinase, Src, Abl, VEGF, and 1ck, and thus be effective in the treatment of diseases associated with other protein kinases.
Compounds of formula I also induce or inhibit apoptosis, a physiological cell death process critical for normal development and homeostasis. Alterations of apoptotic pathways contribute to the pathogenesis of a variety of human diseases. Compounds of formula I, as modulators of apoptosis, will be useful in the treatment of a variety of human diseases with abberations in apoptosis including cancer (particularly, but not limited to follicular lymphomas, carcinomas with p53 mutations, hormone dependent tumors of the breast, prostate and ovary, and precancerous lesions such as familial adenomatous polyposis), viral infections (including but not limited to herpesvirus, poxvirus, Epstein-Barr virus, Sindbis virus and adenovirus), autoimmune diseases (including but not limited to systemic lupus, erythematosus, immune mediated glomerulonephritis, rheumatoid arthritis, psoriasis, inflammatory bowel diseases, and autoimmune diabetes mellitus), neurodegenerative disorders (including but not limited to Alzheimer""s disease, AIDS-related dementia, Parkinson""s disease, amyotrophic lateral sclerosis, retinitis pigmentosa, spinal muscular atrophy and cerebellar degeneration), AIDS, myelodysplastic syndromes, aplastic anemia, ischemic injury associated myocardial infarctions, stroke and reperfusion injury, arrhythmia, atherosclerosis, toxin-induced or alcohol induced liver diseases, hematological diseases (including but not limited to chronic anemia and aplastic anemia), degenerative diseases of the musculoskeletal system (including but not limited to osteoporosis and arthritis), aspirin-sensitive rhinosinusitis, cystic fibrosis, multiple sclerosis, kidney diseases, and cancer pain.
The compounds of this invention may also be useful in combination with known anti-cancer treatments such as radiation therapy or with cytostatic and cytotoxic agents, such as for example, but not limited to, DNA interactive agents, such as cisplatin or doxorubicin; inhibitors of farnesyl protein transferase, such as those described in pending U.S. application Ser. No. 08/802,329, topoisomerase II inhibitors, such as etoposide; topoisomerase I inhibitors, such as CPT-11 or topotecan; tubulin stabilizing agents, such as paclitaxel, docetaxel or the epothilones; hormonal agents, such as tamoxifen; thymidilate synthase inhibitors, such as 5-fluorouracil; and antimetabolites, such as methoxtrexate; antiangiogenic agents, such as angiostatin or endostatin; and kinase inhibitors, such as her2 specific antibodies. The formula I compounds of this invention may also be useful in combination with modulators of p53 transactivation. In addition, the formula I compounds may be used for treating chemotherapy-induced alopecia, chemotherapy-induced thrombocytopenia, chemotherapy-induced leukopenia or mucocitis. In the treatment of chemotherapy-induced alopecia, the formula I compound is preferably topically applied in the form of a medicament such as a gel, solution, dispersion or paste.
If formulated as a fixed dose, such combination products employ the compounds of this invention within the dosage range described below and the other pharmaceutically active agent within its approved dosage range. For example, the cdc2 inhibitor olomucine has been found to act synergistically with known cytotoxic agents in inducing apoptosis (J. Cell Sci., 108, 2897 (1995)). Compounds of formula I may be used sequentially with known anti-cancer or cytotoxic agents when a combination formulation is inappropriate.
cdc2/cyclin B1 kinase activity was determined by monitoring the incorporation of 32P into histone HI. The reaction consisted of 50 ng baculovirus expressed GST-cdc2, 75 ng baculovirus expressed GST-cyclin B1, 1 xcexcg histone HI (Boehringer Mannheim), 0.2 xcexcCi of 32P xcex3-ATP and 25 xcexcM ATP in kinase buffer (50 mM Tris, pH 8.0, 10 mM MgCl2, 1 mM EGTA, 0.5 mM DTT). The reaction was incubated at 30xc2x0 C. for 30 minutes and then stopped by the addition of cold trichloroacetic acid (TCA) to a final concentration of 15% and incubated on ice for 20 minutes. The reaction was harvested onto GF/C unifilter plates (Packard) using a Packard Filtermate Universal harvester, and the filters were counted on a Packard TopCount 96-well liquid scintillation counter (Marshak, D. R., Vanderberg, M. T., Bae, Y. S., Yu, I. J., J. of Cellular Biochemistry, 45, 391-400 (1991), incorporated by reference herein).
cdk2/cyclin E kinase activity was determined by monitoring the incorporation of 32P into the retinoblastoma protein. The reaction consisted of 2.5 ng baculovirus expressed GST-cdk2/cyclin E, 500 ng bacterially produced GST-retinoblastoma protein (aa 776-928), 0.2 xcexcCi 32P xcex3-ATP and 25 xcexcM ATP in kinase buffer (50 mM Hepes, pH 8.0, 10 mM MgCl2, 5 mM EGTA, 2 mM DTT). The reaction was incubated at 30xc2x0 C. for 30 minutes and then stopped by the addition of cold trichloroacetic acid (TCA) to a final concentration of 15% and incubated on ice for 20 minutes. The reaction was harvested onto GF/C unifilter plates (Packard) using a Packard Filtermate Universal harvester, and the filters were counted on a Packard TopCount 96-well liquid scintillation counter.
cdk4/cyclin D1 kinase activity was determined by monitoring the incorporation of 32P in to the retinoblastoma protein. The reaction consisted of 165 ng baculovirus expressed as GST-cdk4, 282 ng bacterially expressed as S-tag cyclin D1, 500 ng bacterially produced GST-retinoblastoma protein (aa 776-928), 0.2 xcexcCi 32P xcex3-ATP and 25 xcexcM ATP in kinase buffer (50 mM Hepes, pH 8.0, 10 mM MgCl2, 5 mM EGTA, 2 mM DTT). The reaction was incubated at 30xc2x0 C. for 1 hour and then stopped by the addition of cold trichloroacetic acid (TCA) to a final concentration of 15% and incubated on ice for 20 minutes. The reaction was harvested onto GF/C unifilter plates (Packard) using a Packard Filtermate Universal harvester, and the filters were counted on a Packard TopCount 96-well liquid scintillation counter (Coleman, K. G., Wautlet, B. S., Morissey, D, Mulheron, J. G., Sedman, S., Brinkley, P., Price, S., Wedster, K. R. (1997) Identification of CDK4 Sequences involved in cyclin D, and p16 binding. J. Biol. Chem. 272,30:18869-18874, incorporated by reference herein).